Phage display selection of anti fungal peptides

ABSTRACT

A method for the identification of peptides having an affinity for the surface of fungi is provided as is a method for the identification of peptides capable of affecting the development of a fungus. Also provided are compositions comprising peptides identified using the method of the present invention. In addition, isolated polynucleotides, vectors, expression cassettes and transformed cells capable of expressing peptides identified by the method of the present invention are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/195,785, filed Apr. 10, 2000, which is hereinincorporated by reference in its entirety for all purposes.

BACKGROUND

[0002] This invention relates to the use of phage display technology toidentify peptides that bind to pathogenic fungi and more particularly topathogenic fungi of the genus Phytophthora. Random peptide phage displaylibraries are constructed using degenerate oligonucleotides. Phageexpressing the peptides on their surface are contacted with fungi atdifferent life stages and those phage that bind are isolated, amplifiedand the peptides identified. Once identified, peptides can be screenedfor anti fungal activity and used to identify and characterize bindingsites on fungi.

[0003] Phytophthora is an economically important disease causingorganism in the United States causing large losses in many agronomicallyimportant crop species. Phytophthora sojae is the second most importantpathogen of soybeans in the United States. (Doupnik, Plant Dis.77:1170-1171, 1993). Phytophthora capsici has a broad host range andmost notably limits production of high-value, solanaceous vegetablecrops. Control of these pathogens is particularly difficult, oftenrequiring treatment of entire fields with biocidal compounds. Althougheffective, increasing concern about the environmental and economic costsof such treatments require the need for alternative control methods.

[0004] Phytophthora species are obligate parasites adapted to long-termsurvival in soil in the absence of host plants. Oospores orchlamydospores exist in low densities in the soil and enable survival ofthe pathogen. In the presence of a susceptible plant, the pathogenprogresses rapidly through a series of finely tuned developmental stepsthat produce cycles of infection and disease. Pathogen development fromoospores or chlamydospores through zoospore release, encystment,germination and infection appear straight-forward at first glance. Yet,the procession of life stages is finely tuned to environmental signals,particularly those signals coming from a host plant.

[0005] Zoospores are the life-stage of greatest importance for dispersalto root infection sites. A major susceptible site is located just behindthe apical meristem of the root where cells are elongating. Exudatesreleased from elongating cells serve as signals that direct chemotacticmovement of zoospores toward the site (Carlile, in Phytophthora: ItsBiology, Taxonomy, Ecology and Pathology, Erwin et al., eds., APS Press,1983; Deacon and Donaldson, Mycol. Res. 97:1153-1171, 1993). Thezoospore chemotactic response varies with the composition of rootexudates and is species specific. For example, zoospores of P. capsici,P. cactorum, and other species are attracted to an array of sugars andamino acids (Hickman, Phytopathology, 60:1128-1135, 1970; Khew andZentmyer, Phytopathology, 63:1511-1517, 1973), but zoospores of P. sojaeare attracted to specific isoflavonoid compounds (Norris et al., PlantPhysiol., 117:1171-1178, 1998). Although the precise mechanism ofchemoattraction is not known, Deacon and Donaldson (Mycol. Res.,97:1153-1171, 1993) and Carlile (in Phytophthora: Its Biology, Taxonomy,Ecology and Pathology, Erwin et al., eds., APS Press, 1983) summarizedexperiments that suggested the involvement of chemoreceptors on thezoospore surface.

[0006] Zoospores encyst as they approach the root surface in response toenvironmental signals. Encystment of zoospores of P. palmivora and otherPhytophthora species, for example, can be influenced by local calciumion concentrations (Griffith et al., Arch. Microbiol., 149:565-571,1988; Warburton and Deacon, Fungal Genetics Biol., 25:54-62, 1998).Encystment can also be induced by high concentration of chemoattractantsor by root cell wall components. For example, zoospores of P. sojaeencyst in the presence of high concentrations of soybean isoflavonoidcompounds (Morris and Ward, Physiol. Mol. Plant Pathol., 40:17-22,1992). In contrast, zoospores of Pythium aphanidermatum encysted when incontact with fucosyl and galactosyl residues from cell surfaces of cressroots (Longman and Callow, Physiol. Mol. Plant Pathol., 30:139-150,1987; Estrada-Garcia et al., J. Exp. Bot. 41:693-699, 1990). Deacon andDonaldson (Mycol. Res., 97:1153-1171, 1993) noted that encystment in thepresence of high concentrations of attractants would be deleterious toinfection potential, and thus selected against over time. Theysuggested, however, that attractants at the root surface may besufficiently concentrated to predispose zoospores to encyst aftercontact with root surface residues.

[0007] When in contact with a root, zoospores encyst with a specificorientation so that a germ tube emerges toward the root. If zoosporesencyst before contact with the root, the germ tubes will emerge in anyorientation and must re-orient in order to locate the root and infectthe plant. Cell surface receptors on the germ tube are thought to beinvolved in this root-orientation process. Morris et al. (PlantPhysiol., 117:1171-1178, 1998), for example, demonstrated an orientedresponse of P. sojae germling growth to low, nontoxic concentrations ofisoflavonoid compounds derived from soybeans. Zentmyer (Science,133:1595-1596, 1961) reported hyphal orientation of P. cinnamomi towardhost roots, but the nature of the attractant compound(s) was notdefined.

[0008] After infection, hyphae grow through plant tissue intercelluarlyand/or intracellularly depending on the species of pathogen (Stossel etal., Can. J. Bot., 58:2594-2601, 1980; Coffey and Wilson, inPhytophthora: Its Biology, Taxonomy, Ecology and Pathology, Erwin etal., eds., APS Press, 1983; Enkerli et al., Can. J. Bot., 75:1493-1508,1997; Hardham and Mitchell, Fungal Gen. Biol., 24:252-284, 1998; Murdochand Hardham, Protoplasma, 201:180-193, 1998). Haustoria are formed bysome Phytophthora species, including P. infestans (Coffey and Wilson, inPhytophthora: Its Biology, Taxonomy, Ecology and Pathology, Erwin etal., eds., APS Press, 1983), P. capsici (Jones et al., Phytopathology,64:1084-1090, 1974), and P. sojae (Stössel et al., Can. J. Bot.,58:2594-2601, 1980). Both hyphae and haustoria establish close contactwith host cell walls and membranes. Presumably, cell surface receptorsare important in sensing plant signals, although direct evidence islacking. Indirect evidence of cell surface receptors comes fromobservations such as the occurrence of vesicles in the distal portion ofhaustoria (Coffey and Wilson, in Phytophthora: Its Biology, Taxonomy,Ecology and Pathology, Erwin et al., eds., APS Press, 1983). Heath (Can.J. Bot., 73(Suppl.):S131-S139, 1995) discussed the possible events offungal hyphal tip growth involving communication with the surroundingenvironment via ion channel and vesicle functions.

[0009] As discussed above, evidence points to the prominence of cellsurface receptors in triggering behavioral and developmental steps ofPhytophthora. Cells surface receptors, therefore, may provide a meansfor disrupting pathogen development and so infectivity. Delay ordisruption of development can have a substantial impact, since zoosporeshave only a limited time to locate, contact, and penetrate an infectionsite that is effectively moving with the growing root tip. This timelimitation results from the changing susceptibility of the root tissues,since as the tissues in the elongation region mature, they becomesignificantly less susceptible to infection (English and Mitchell,Phytopathology, 78:1478-1483, 1988).

[0010] “Fusion phage” are filamentous bacteriophage vectors in whichforeign peptides and proteins are cloned into a phage coat gene anddisplayed as part of a phage coat protein. The commonly used coat genesfor the production of fusion phage are the pVIII gene and the pIII gene.About 3900 copies of pVIII make up the major portion of the tubularvirion protein coat. Each pVIII coat protein lies at a shallow angle tothe long axis of the virion, with its C-terminus buried in the interiorclose to the DNA and its N-terminus exposed to the external environment.Five copies of the pIII coat protein are located at the terminal end ofeach virion and are involved in attachment of the phage to pIII of E.coli and for virus reassembly after infection and replication. Peptidesdisplayed as part of pVIII are constrained in the matrix of theirdisplay on the virion coat. In contrast, peptides displayed as part ofpIII are more flexible due to the terminal position of the pIIIproteins. Specific phage can be constructed to display peptides of sixto 15 amino acids in length. Insertion of random or degenerateoligonucleotides into the coat protein genes allows the production ofphage displayed random peptide libraries. A typical display librarycontains 10 to 100 copies of as many as 10⁸ random sequence peptides.Thus, phage display is useful for screening for rare peptides withdesired binding characteristics.

[0011] Phage-displayed random peptide libraries have been used forisolating ligands to cell surface receptors on mammalian cells. Forexample, peptides have been isolated from phage-displayed libraries thatbind the transmembrane integrin glycoproteins involved incell-extracellular matrix and cell-cell interactions (O'Neil et al,Proteins, 14:509-515, 1992; Smith et al., J. Biol. Chem.,269:32788-32795, 1994; Healy et al., Biochemistry, 34:3948-3955, 1995).The phage displayed peptides specifically blocked cell adhesion todefined extracellular molecules and other cells (Koivunen et al., J.Biol. Chem., 268:20205-20210, 1993; Koivunen et al., J. Cell Biol.,124:373-380, 1994; Healy et al., Biochemistry, 34:3948-3955, 1995;Pasqualini et al., Nature Biotech., 15: 542-547, 1997). Phage-displayedrandom peptide libraries have also been used to select peptides thatdistinguish between brain and kidney tissue (Pasqualini and Ruoslahti,Nature, 380:364-366, 1996). In vivo, affinity-selection ofphage-displayed random peptides has also been used to select peptidesthat bind selectively to endothelial cells of blood vessels of specifictumor tissues (Pasqualini et al., Nature Biotech., 15: 542-547, 1997).When these peptides were fused to an anti-cancer drug and injected intotumor-bearing mice, the peptides successfully targeted the drug to tumorblood vessels and deterred progressive tumor development (Arap et al.,Science, 279:377-380, 1998).

[0012] Phage-display methods have been applied to plant pathogens inonly very limited circumstances. Phage display methods have been usedalmost exclusively to identify antibodies for plant virus diagnosis(Susi et al., Phytopathology, 88:230-233, 1998; Ziegler et al.,Phytopathology, 88:1302-1305, 1998; Griep et al., J. Plant Pathol.,105:147-156 1999; Toth et al., Phytopathology, 89:1015-1021, 1999).Phage display was used in a single instance to select antibodies withaffinity to surface-exposed epitopes on germlings and spores ofPhytophthora infestans (Gough et al., J. Immunol. Methods, 228:97-108,1999). Isolated phage-displayed, single-chain variable fragment (Fv)antibody fragments were not assessed for their potential to influencespore or germling behavior. Antibodies were tested for their antifungalactivity with sporangia, but were found to have no detectable antifungalactivity.

[0013] What is needed, therefore, is a rapid and efficient method forscreening peptides for specific binding to plant pathogens. Onceidentified, the peptides can be further evaluated for their ability toprevent infection of plants by the pathogen, and suitable peptides canbe applied directly to the plant, used to treat the soil or,alternatively, sequences encoding the peptides can be introduced in theplants to confer immunity against the pathogen. In this manner,economical and environmentally safe and effective methods of controllingplant pathogens can be developed. The present invention meets this need.

SUMMARY

[0014] Among the several aspects of the present invention, therefore, isto provide a method for identifying peptides having an affinity for thesurface of a plant pathogen comprising, constructing a library of randompeptides by providing degenerate oligonucleotides encoding peptides;inserting the oligonucleotides into an appropriate vector that expressesthe encoded peptides on its surface and is capable of transfecting ahost cell; and transfecting an appropriate host cell with the vector toamplify the vector in a infectious form to create a library of peptideson the vector. The vector expressing the peptide library is thencontacted with a target pathogen and allowed to bind to the pathogen.Unbound vector is removed and vector that has bound to the pathogeneluted. The eluted vector is then amplified in a suitable host cell andthe inserted oligonucleotides isolated. The oligonucleotides are thensequenced by any suitable method and the amino acid sequence of thepeptides deduced from the sequence of the oligonucleotides.

[0015] Another aspect of the invention provides an antifungalcomposition comprising at least one peptide selected from the groupconsisting of ADPPRTVST (SEQ ID NO: 7), ADRPSMSPT (SEQ ID NO: 8),ADITDPMGA (SEQ ID NO: 20), AVGTHTPDS (SEQ ID NO: 21), AVSPNVHDG (SEQ IDNO: 22), LTRCLVSTEMAARRP (SEQ ID NO: 24), EFRKNYPSAAPLIPR (SEQ ID NO:31), LFXCYPPCTYSYCLS (SEQ ID NO: 33), and AAPDLQDAM (SEQ ID NO: 4).

[0016] Still another aspect of the present invention is a recombinantnucleotide comprising a sequence encoding a peptide selected from thegroup consisting of ADPPRTVST (SEQ ID NO: 7), ADRPSMSPT (SEQ ID NO: 8),ADITDPMGA (SEQ ID NO: 20), AVGTHTPDS (SEQ ID NO: 21), AVSPNVHDG (SEQ IDNO: 22), LTRCLVSTEMAARRP (SEQ ID NO: 24), EFRKNYPSAAPLIPR (SEQ ID NO:31), LFXCYPPCTYSYCLS (SEQ ID NO: 33), and AAPDLQDAM (SEQ ID NO: 4).

[0017] Yet another aspect of the present invention is a recombinantvector comprising a nucleotide sequence encoding a peptide selected fromthe group consisting of ADPPRTVST (SEQ ID NO: 7), ADRPSMSPT (SEQ ID NO:8), ADITDPMGA (SEQ ID NO: 20), AVGTHTPDS (SEQ ID NO: 21), AVSPNVHDG (SEQID NO: 22), LTRCLVSTEMAARRP (SEQ ID NO: 24), EFRKNYPSAAPLIPR (SEQ ID NO:31), LFXCYPPCTYSYCLS (SEQ ID NO: 33), and AAPDLQDAM (SEQ ID NO: 4).

[0018] A further aspect of the present invention is a cell transformedwith a vector comprising a nucleotide sequence encoding a peptideselected from the group consisting of ADPPRTVST (SEQ ID NO: 7),ADRPSMSPT (SEQ ID NO: 8), ADITDPMGA (SEQ ID NO: 20), AVGTHTPDS (SEQ IDNO: 21), AVSPNVHDG (SEQ ID NO: 22), LTRCLVSTEMAARRP (SEQ ID NO: 24),EFRKNYPSAAPLIPR (SEQ ID NO: 31), LFXCYPPCTYSYCLS (SEQ ID NO: 33), andAAPDLQDAM (SEQ ID NO: 4).

[0019] In still another aspect is provided an expression cassettecomprising as operatively linked components, a promoter; a nucleotidesequence encoding a peptide selected from the group consisting ofADPPRTVST (SEQ ID NO: 7), ADRPSMSPT (SEQ ID NO: 8), ADITDPMGA (SEQ IDNO: 20), AVGTHTPDS (SEQ ID NO: 21), AVSPNVHDG (SEQ ID NO: 22),LTRCLVSTEMAARRP (SEQ ID NO: 24), EFRKNYPSAAPLIPR (SEQ ID NO: 31),LFXCYPPCTYSYCLS (SEQ ID NO: 33), and AAPDLQDAM (SEQ ID NO: 4); and atranscription termination signal sequence.

[0020] An additional aspect provides, a recombinant plant comprising anexpression cassette comprising a promoter; a nucleotide sequenceencoding a peptide selected from the group consisting of ADPPRTVST (SEQID NO: 7), ADRPSMSPT (SEQ ID NO: 8), ADITDPMGA (SEQ ID NO: 20),AVGTHTPDS (SEQ ID NO: 21), AVSPNVHDG (SEQ ID NO: 22), LTRCLVSTEMAARRP(SEQ ID NO: 24), EFRKNYPSAAPLIPR (SEQ ID NO: 31), LFXCYPPCTYSYCLS (SEQID NO: 33), and AAPDLQDAM (SEQ ID NO: 4); and a transcriptiontermination signal sequence.

[0021] Another aspect provides a method for characterization of peptideshaving an affinity for the surface of plant pathogens comprisingproviding a library of random peptides made by providing degenerateoligonucleotides encoding peptides; inserting the oligonucleotides intoan appropriate vector that expresses the peptides on its surface and iscapable of transfecting a host cell; and transfecting an appropriatehost cell with the vector to amplify the vector in an infectious form tocreate a library of peptides on the vector. The vector expressing thepeptide library is then contacted with a plant pathogen of interest andthe vector allowed to bind to the pathogen. After binding, the unboundvector is removed and the bound vector eluted from the pathogen. Theeluted vector is amplified in a suitable host cell and the insertedoligonucleotides in the eluted vectors isolated. The peptides encoded bythe oligonucleotides are then produced, contacted with plant pathogensof interest, and the effect on infectivity observed. In one embodiment,the isolated oligonucleotides are sequenced, the amino acid sequence ofthe peptides deduced from the nucleotide sequence, and the peptidesproduced by chemical synthesis. In another embodiment, peptides areproduced by inserting the isolated oligonucleotides into an expressionvectors which is used to transform a suitable host cell. The transformedhost cells are then maintained under conditions suitable for expressionof the peptides.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims and accompanying figures where:

[0023]FIG. 1 shows the organization of f8 peptide sequences into sixfamilies. On the left is the dendogram and on the right are the namesand sequences of the peptides.

[0024]FIG. 2 shows encystment of P. capsici zoospores in response tocontact with the indicated f8 phage-displayed peptides at the variousconcentrations given. Percentage values represent the means of twoexperiments. The percentage encystment for the control zoosporepopulation that contained no phage varied between 0 and 10%.

[0025]FIG. 3 shows encystment of P. capsici zoospores in response tocontact with the indicated f8 and f88-4 phage-displayed peptides at thevarious concentrations given.

[0026]FIG. 4 shows binding of the indicated phage-displayed peptides toP. capici zoospsores. Values represent the mean of three experiments.

[0027]FIG. 5 shows the binding specificity of the indicatedphage-displayed peptides to P. capici. Values represent the mean ofthree experiments.

[0028]FIG. 6 is a map of plasmid pJE-7. AOX-P is the alcohol oxidasepromoter, Mat-α is the mat-alpha secretory sequence, CKX1 is thecytokinin oxidase 1 sequence, Pc87 is an exemplary peptide of thepresent invention, and AOX-TT is the alcohol oxidase terminationsequence. Below the map is the double stranded sequence encoding Pc87showing the (+) strand (5′ AG CTA GCA GAT AGA CCA TCA ATG TCA CCA ACATAG T 3′, SEQ ID NO: 46) and the (−) strand (5′ CT AGA CTA TGT TGG TGACAT TGA TGG TCT ATC TGC T 3′, SEQ ID NO: 47).

[0029]FIG. 7 shows the amino acid sequence (SEQ ID NO: 48) of theexemplary insert contained in pJE-7 where the underlined nucleotides arethe mat-alpha secretory sequence (cleavage by the Kex2 enzyme occursafter the last underlined arginine), followed by the cytokinin oxidiase1 sequence. The two double-underlined amino acids (KL) were added to aidin construction of the fusion protein. The amino acid sequence of theexemplary peptide Pc87 is shown in bold type.

DEFINITIONS

[0030] “Secretion sequence” means a sequence that directs newlysynthesized secretory or membrane proteins to and through membranes ofthe endoplasmic reticulum, or from the cytoplasm to the periplasm acrossthe inner membrane of bacteria, or from the matrix of mitochondria intothe inner space, or from the stroma of chloroplasts into the thylakoid.Fusion of such a sequence to a gene that is to be expressed in aheterologous host ensures secretion of the recombinant protein from thehost cell.

[0031] “Germling” means a newly germinated cyst (5-8 hr postgermination) that bears an emergent germ tube.

[0032] “TBS” means Tris-buffered saline (50 mM Tris-HCl, pH 7.5, 150 mMNaCl).

[0033] A “recombinant polynucleotide” means a polynucleotide that isfree of one or both of the nucleotide sequences which flank thepolynucleotide in the naturally-occurring genome of the organism fromwhich the polynucleotide is derived. The term includes, for example, apolynucleotide or fragment thereof that is incorporated into a vector orexpression cassette; into an autonomously replicating plasmid or virus;into the genomic DNA of a prokaryote or eukaryote; or that exists as aseparate molecule independent of other polynucleotides. It also includesa recombinant polynucleotide that is part of a hybrid polynucleotide,for example, one encoding a polypeptide sequence.

[0034] “IPTG” is isopropylthiogalactoside.

[0035] “TU” means transducing unit.

[0036] “NAP buffer” is 80 mM NaCl, 50 mM NH₄H₂PO₄, pH adjusted to 7.0with NH₄OH.

[0037] “NZY-Tc” is a bacterial growth medium containing 1% NZ amine A (atyptone-type medium; Humko-Sheffield Chemical, Norwich, N.Y.), 0.5%yeast extract, 0.5% NaCl, pH 7.0 adjusted with NaOH.

[0038] “PCR” means polymerase chain reaction.

[0039] As used herein “polynucleotide” and “oligonucleotide” are usedinterchangeably and refer to a polymeric (2 or more monomers) form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides. Although nucleotides are usually joined byphosphodiester linkages, the term also includes polymeric nucleotidescontaining neutral amide backbone linkages composed of aminoethylglycine units. This term refers only to the primary structure of themolecule. Thus, this term includes double- and single-stranded DNA andRNA. It also includes known types of modifications, for example, labels,methylation, “caps”, substitution of one or more of the naturallyoccurring nucleotides with an analog, internucleotide modifications suchas, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.),those containing pendant moieties, such as, for example, proteins(including for e.g., nucleases, toxins, antibodies, signal peptides,poly-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotide. Polynucleotidesinclude both sense and antisense strands.

[0040] “Sequence” means the linear order in which monomers occur in apolymer, for example, the order of amino acids in a polypeptide or theorder of nucleotides in a polynucleotide.

[0041] “Peptide” and “Protein” are used interchangeably and mean acompound that consists of two or more amino acids that are linked bymeans of peptide bonds.

[0042] “Recombinant protein” means that the protein, whether comprisinga native or mutant primary amino acid sequence, is obtained byexpression of a gene carried by a recombinant DNA molecule in a cellother than the cell in which that gene and/or protein is naturallyfound. In other words, the gene is heterologous to the host in which itis expressed. It should be noted that any alteration of a gene,including the addition of a polynucleotide encoding an affinitypurification moiety, makes that gene unnatural for the purposes of thisdefinition, and thus that gene cannot be “naturally” found in any cell.

[0043] A “non-immunoglobulin peptide” means a peptide which is not animmunoglobulin, a recognized region of an immunoglobulin, or contains aregion of an immunoglobulin. For example, a single chain variable regionof an immunoglobulin would be excluded from this definition.

[0044] “Substantially pure” or “substantially purified” means that thesubstance is free from other contaminating proteins, nucleic acids, andother biologicals derived from the original source organism. Purity maybe assayed by standard methods, and will ordinarily be at least about40% pure, more ordinarily at least about 50% pure, generally at leastabout 60% pure, more generally at least about 70% pure, often at leastabout 75% pure, more often at least about 80% pure, typically at leastabout 85% pure, more typically at least about 90% pure, preferably atleast about 95% pure, more preferably at least about 98% pure, and ineven more preferred embodiments, at least 99% pure. The analysis may beweight or molar percentages, evaluated, e.g., by gel staining,spectrophotometry, or terminus labeling etc.

DETAILED DESCRIPTION

[0045] The following detailed description is provided to aid thoseskilled in the art in practicing the present invention. Even so, thisdetailed description should not be construed to unduly limit the presentinvention as modifications and variations in the embodiments discussedherein can be made by those of ordinary skill in the art withoutdeparting from the spirit or scope of the present inventive discovery.

[0046] All publications, patents, patent applications, databases andother references cited in this application are herein incorporated byreference in their entirety as if each individual publication, patent,patent application, database or other reference were specifically andindividually indicated to be incorporated by reference.

[0047] In one embodiment peptide libraries are constructed by theinsertion of nucleic acid sequences encoding peptides of six to 15 aminoacids in length into suitable vectors, although sequences encodinglonger peptides can be used. The peptides encoded by the nucleotides canbe completely random in nature or can be constrained in theircomposition to meet structural or functional requirements. For example,and without limitation, a cysteine bridge can be inserted into thepeptide. In one embodiment, the nucleic acid sequence does not encode animmunoglobulin (antibody) or a recognized immunoglobulin region such asa variable region. Any vector which will express the insertedoligonucleotides can be used. Preferably a vector is used which willresult in expression of the peptide library on the surface of a cell orvirus or on the surface of an intracellular compartment or organelle ofa cell or virus. In this manner, the expressed peptides will beavailable to interact with potential target molecules or cells that comein contact with the surface containing the peptides. As will be apparentto one of ordinary skill in the art, if the peptides are expressed onthe surface of intracellular compartments or organelles, the potentialtarget must also reside intracellularly or the organelle orintracellular compartment must be exposed to the external environmentby, for example, lysis of the cell.

[0048] Methods of producing oligonucleotides and inserting them intovectors are well known to those of ordinary skill in the art and willonly be briefly reviewed herein. Most commonly, oligonucleotides aresynthesized on a solid support using the phosphite triester method ofBeaucage and Caruthers (Tetrahedron Lett. 22:1859-1862, 1981; also see,U.S. Pat. Nos. 4,973,679 and 4,458,066). Numerous solid supports areavailable including controlled pore glass beads, polystyrene copolymers,silica gel and cellulose paper. The preparation of an oligonucleotidebegins with the linkage of the 3′-hydroxyl group of the first nucleosideto the solid support. Solid supports containing nucleotides areavailable from commercial sources. The oligonucleotide is synthesizedfrom the 3′ to 5′ direction and the chain is elongated by nucleophilicattack of the 5′-hydroxyl of the immobilized oligonucleotide on theactivated 3′ phosphate or phosorphramidite of a soluble 5′-protectedbuilding block. The intermediate dinucleoside phosphite formed must nextbe oxidized to the more stable phosphate before chain extension. Theprocess is repeated until the desired number of nucleotides has beenadded. Automated devices are commercially available for the synthesis ofoligonucleotides. In addition, numerous commercial vendors providecustom oligonucleotide synthesis services.

[0049] Any vector system capable of expressing the peptides of thepeptide library may be used in the practice of the present invention andnumerous vector systems are known in the art (See e.g., Wilson andFindlay, Can. J. Microbiol., 44:313-329, 1998). When the peptide isdisplayed as part of pVIII, suitable phage systems include type 8, type88, and type 8+8. When pIII is utilized suitable phage systems includetype 3, type 33 and type 3+3. When the peptide is inserted into pVI,suitable phage systems included type 6, type 66 and type 6+6. Inaddition, phage T7 and phage 8 vector systems can be used. In onepreferred embodiment, the peptides of the library are expressed fused toa coat protein of a filamentous bacteriophage so that the peptides areexpressed on the surface of the virion and so are available to interactwith target molecules or cell surface receptors. In one preferredembodiment, the f8-1 library is used in which random 8-mer peptides arefused to the pVIII coat protein. In another preferred embodiment, thef88-4 library is used in which random 15-mer peptides are fused to thepVIII coat protein. Phage in the f88-4 library display peptides withoutbias toward the occurrence of any amino acid. Phage in the f8-1 libraryare unbiased with the exception of alanine at the first position and oneof four residues at the second position. All other positions arerandomly occupied by any amino acid.

[0050] Methods for production of the f8-1 phage-displayed peptidelibrary have been described previously (See, Petrenko et al., Prot.Engineering, 9:797-801, 1996 and references cited therein). The librarydisplays foreign peptides on every copy of the 3900 copies of major coatprotein pVIII. Peptide expression need not be induced by IPTG. Thedegenerate oligonucleotide used for the 8-amino acid insert is: GCA GNN(NNN)₇, where N is any nucleotide. Therefore, the first amino acid is analanine (A) and the second amino acid is a valine (V), alanine (A),aspartate (D), glutamate (E) or glycine (G). The remainder of the aminoacids in the peptide are completely randomized.

[0051] Likewise, methods for the production of the f88-4 phage-displayedpeptide library have also been previously described (Zhong et al., J.Biol. Chem. 269:24183-24188, 1994; Smith and Scott, Methods inEnzymology, 217:228-257, 1993; Smith, Gene, 128:1-2, 1993 and referencescited therein). This library displays 15-amino acid foreign peptides on150 to 300 copies of major coat protein pVIII. The remainder of the 3900copies of the pVIII subunits are derived from the wild type pVIII. Thephage genome thus bears two pVIII genes encoding two different types ofpVIII molecules. One pVIII is the recombinant displaying the foreign15-mer peptide, while the other is the wild-type pVIII normally presenton the phage. Expression of the recombinant pVIII gene is driven by theIPTG inducible tac promoter/operator. Because of the presence of twopVIII genes, the f88 virion consists of a mosaic pattern of wild-typeand recombinant pVIII subunits.

[0052] The oligonucleotide sequence used for the 15-mer amino acidinserts is (NNK)₁₅, where N is A, T, C, or G and K designates G or T.Thus, the region surrounding the 15-mer insert is:LVPMLSFA(X)₁₅PAEGDDPAKA (SEQ ID NO: 1), where X is any amino acidencoded by the codon NNK.

[0053] The phage particles can be used to screen the random peptidesexpressed on the virion for their ability to bind to compounds and cellsof interest. In one preferred embodiment, the phage-displayed peptidelibrary is used to screen for peptides that bind to plant pathogens. Inanother preferred embodiment the peptides are screened for their abilityto bind to pathogenic fungi. In still another preferred embodiment,phage-displayed peptides are screened for their ability to bind tomembers of the genus Phytophthora. When examining pathogens with morethan a single life stage, it is preferable that each life stage beexamined, since significant differences in the number, types andaffinity of binding sites can occur with changes in developmentalstages.

[0054] For example, when examining members of the genus Phytophthora,approximately 10⁵ to 10⁶ organisms are mixed with approximately 10⁸ to10⁹ phage-displayed peptides and incubated for a time sufficient toallow binding. It will be apparent to those of ordinary skill in the artthat depending on factors such as the species of pathogen, the phage andthe peptide, that other concentrations of organisms and displayedpeptides can be used within the scope of the present invention. In somecases, it may be desirable to pre-incubate the displayed peptides withother life stages of the same organism in order to identify thosepeptides that bind only to a specific life stage. After incubation, theorganism is subject to multiple washes in order to remove unbound andweakly bound peptides. In the case of Phytophthora zoospores, washing isdone using a solution of approximately 50 mM LiCl. After washing, boundphage-displayed peptides are eluted, preferrably at low pH, and theeluted phage amplified in a suitable host. In one embodiment, the hostis starved K91 E. coli. Methods for the amplification of bacteriophagein E. coli are well known in the art and can be found, for example, inSmith and Scott, Methods in Enzymology, 217:228-257, 1993; Ausubel etal. eds., Short Protocols in Molecular Biology, 2nd ed., Wiley & Sons,1995; and Sambrook et al., Molecular Cloning, Cold Spring HarborLaboratory Press, 1989. In one embodiment, the screening procedure isrepeated at least once in order to enrich high-affinity phage displayedpeptides. In another embodiment, the screening process is repeated threetimes.

[0055] Once phage displayed high affinity peptides are identified, thephage are amplified, preferrably in E. coli, and the phage DNA isolatedusing standard methods such as those found in, for example Smith andScott, Methods in Enzymology, 217:228-257, 1993; Ausubel et al. eds.,Short Protocols in Molecular Biology, 2nd ed., Wiley & Sons, 1995; andSambrook et al., Molecular Cloning, 2nd ed., Cold Spring HarborLaboratory Press, 1989. Once the phage DNA has been isolated, theinserted oligonucleotides can be cleaved from the DNA using the samerestriction enzymes used to insert the oligonucleotides, and therestriction enzyme fragments separated from the remainder of the DNA.The oligonucleotides can then be sequenced using any standard method.Sequencing can be carried out by any suitable method, for example,dideoxy sequencing (Sanger et al., Proc. Natl. Acad. Sci. USA,74:5463-5467, 1977), chemical sequencing (Maxam and Gilbert, Proc. Natl.Acad. Sci. USA, 74:560-564, 1977) or any variation thereof, includingthe use of automatic sequencers. In one embodiment, sequencing isaccomplished using an ABI Prism 377 automated sequencer (AppliedBiosystems, Foster City, Calif.). Once the sequence of theoligonucleotides is known, the amino acid sequences of the peptidesencoded can be readily deduced using the genetic code.

[0056] High-affinity binding, phage displayed peptides can be furtherscreened for their ability to alter the development, growth and/orinfectivity of pathogens. In this embodiment, phage-displayed peptidesare incubated with a target pathogen for a time sufficient to allowbinding. Following binding, the pathogen is observed for alterations inits development or ability to infect a host. In one embodiment,approximately 200 zoospores of a member of the genus Phytophthora arecombined with a phage-displayed peptide in distilled water in two-foldserial dilutions at constant volume in petri dishes. The range of phageconcentrations can vary, but generally ranges between 1 to 10×10⁹virion/μl. A negative control containing no phage is included in eachscreening. After an incubation period of usually about 20 minutes atroom temperature, the number of zoospores encysted at each phageconcentration is determined. Using this method it is possible torationally select peptides of defined character and evaluate them forspecies- and life stage-specific induction of receptor-mediatedfunctional responses, such a zoospore encystment. Peptides found tointerfere with the development of a pathogen can be used to prevent orlimit infection of a host with the pathogen.

[0057] The present method can also be used to characterize peptidebinding receptors on the surface of plant pathogens. In this embodiment,peptide displaying phage that have been labeled (test phage) areincubated with cells of different organisms and at different stages ofdevelopment. The relative binding affinity of the labeled phage can thenbe determined by competitive binding and Scatchard analysis. In acompetitive binding analysis, a constant concentration of test phage isallowed to bind to a target pathogen and then unlabeled challenge phageis added over a range of concentrations. The challenge phage may be thesame as the test phage or it may be different. The target pathogen isthen washed to remove non-specifically or weakly bound phage and theamount of test phage bound is determined by measuring the amount oflabel present on the target cells. The degree of competition can bemeasured as the concentration of challenge phage required to inhibittest phage binding by 50% (IC₅₀). Results from competition assays can beuse to determine changes in the number, type and affinity of cellsurface receptors over time.

[0058] Within the scope of the present invention are recombinantoligonucleotides discovered by the method of the present inventionencoding peptides having antifungal activity. These recombinantoligonucleotides can be used to produce recombinant polynucleotideswhich are commonly used as cloning or expression vectors although otheruses are possible. A cloning vector is a self-replicating DNA moleculethat serves to transfer a DNA segment into a host cell. The three mostcommon types of cloning vectors are bacterial plasmids, phages, andother viruses. An expression vector is a cloning vector designed so thata coding sequence inserted at a particular site will be transcribed andtranslated into a protein.

[0059] Both cloning and expression vectors contain nucleotide sequencesthat allow the vectors to replicate in one or more suitable host cells.In cloning vectors, this sequence is generally one that enables thevector to replicate independently of the host cell chromosomes, and alsoincludes either origins of replication or autonomously replicatingsequences. Various bacterial and viral origins of replication are wellknown to those skilled in the art and include, but are not limited tothe pBR322 plasmid origin, the 2μ plasmid origin, and the SV40, polyoma,adenovirus, VSV and BPV viral origins.

[0060] The oligonucleotide sequences of the present invention may beused to produce antifungal peptides by the use of recombinant expressionvectors containing the oligonucleotide sequence. Suitable expressionvectors include chromosomal, non-chromosomal and synthetic DNAsequences, for example, SV 40 derivatives; bacterial plasmids; phageDNA; baculovirus; yeast plasmids; vectors derived from combinations ofplasmids and phage DNA; and viral DNA such as vaccinia, adenovirus, fowlpox virus, and pseudorabies. In addition, any other vector that isreplicable and viable in the host may be used.

[0061] The nucleotide sequence of interest may be inserted into thevector by a variety of methods. In the most common method the sequenceis inserted into an appropriate restriction endonuclease site(s) usingprocedures commonly known to those skilled in the art and detailed in,for example, Sambrook et al., Molecular Cloning, A Laboratory Manual,2nd ed., Cold Spring Harbor Press, (1989) and Ausubel et al., ShortProtocols in Molecular Biology, 2nd ed., John Wiley & Sons (1992).

[0062] In an expression vector, the sequence of interest is operablylinked to a suitable expression control sequence or promoter recognizedby the host cell to direct mRNA synthesis. Promoters are untranslatedsequences located generally 100 to 1000 base pairs (bp) upstream fromthe start codon of a structural gene that regulate the transcription andtranslation of nucleic acid sequences under their control. Promoters aregenerally classified as either inducible or constitutive. Induciblepromoters are promoters that initiate increased levels of transcriptionfrom DNA under their control in response to some change in theenvironment, e.g. the presence or absence of a nutrient or a change intemperature. Constitutive promoters, in contrast, maintain a relativelyconstant level of transcription.

[0063] A nucleic acid sequence is operably linked when it is placed intoa functional relationship with another nucleic acid sequence. Forexample, DNA for a presequence or secretory leader is operatively linkedto DNA for a polypeptide if it is expressed as a preprotein whichparticipates in the secretion of the polypeptide; a promoter is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,operably linked sequences are contiguous and, in the case of a secretoryleader, contiguous and in reading phase. Linking is achieved by ligationat restriction enzyme sites. If suitable restriction sites are notavailable, then synthetic oligonucleotide adapters or linkers can beused as is known to those skilled in the art. Sambrook et al., MolecularCloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, (1989)and Ausubel et al., Short Protocols in Molecular Biology, 2nd ed., JohnWiley & Sons (1992).

[0064] Common promoters used in expression vectors include, but are notlimited to, LTR or SV40 promoter, the E. coli lac or trp promoters, andthe phage lambda PL promoter. Useful inducible plant promoters includeheat-shock promoters (Ou-Lee et al. (1986) Proc. Natl. Acad. Sci. USA83: 6815; Ainley et al. (1990) Plant Mol. Biol. 14: 949), anitrate-inducible promoter derived from the spinach nitrite reductasegene (Back et al. (1991)Plant Mol. Biol. 17: 9), hormone-induciblepromoters (Yamaguchi-Shinozaki et al. (1990) Plant Mol. Biol. 15: 905;Kares et al. (1990) Plant Mol. Biol. 15: 905), and light-induciblepromoters associated with the small subunit of RuBP carboxylase and LHCPgene families (Kuhlemeier et al. (1989) Plant Cell 1: 471; Feinbaum etal. (1991) Mol. Gen. Genet. 226: 449; Weisshaar et al. (1991) EMBO J.10: 1777; Lam and Chua (1990) Science 248: 471; Castresana et al. (1988)EMBO J. 7: 1929; Schulze-Lefert et al. (1989) EMBO J. 8: 651). Otherpromoters known to control the expression of genes in prokaryotic oreukaryotic cells can be used and are known to those skilled in the art.Expression vectors may also contain a ribosome binding site fortranslation initiation, and a transcription terminator. The vector mayalso contain sequences useful for the amplification of gene expression.

[0065] Expression and cloning vectors can, and usually do, contain aselection gene or selection marker. Typically, this gene encodes aprotein necessary for the survival or growth of the host celltransformed with the vector. Examples of suitable markers includedihydrofolate reductase (DHFR) or neomycin resistance for eukaryoticcells and tetracycline or ampicillin resistance for E. coli. Selectionmarkers in plants include resistance to bleomycin, gentamycin,glyphosate, hygromycin, kanamycin, methotrexate, phleomycin,phosphinotricin, spectinomycin, streptomycin, sulfonamide andsulfonylureas. Maliga et al., Methods in Plant Molecular Biology, ColdSpring Harbor Press, 1995, p. 39.

[0066] In addition, expression vectors can also contain marker sequencesoperatively linked to a nucleotide sequence for a protein that encode anadditional protein used as a marker. The result is a hybrid or fusionprotein comprising two linked and different proteins. The marker proteincan provide, for example, an immunological or enzymatic marker for therecombinant protein produced by the expression vector. Suitable markersinclude, but are not limited to, alkaline phosphatase (AP), myc,hemagglutinin (HA), β-glucuronidase (GUS), luciferase, and greenfluorescent protein (GFP).

[0067] The polynucleotide sequences of the present invention can also bepart of an expression cassette that at a minimum comprises, operablylinked in the 5′ to 3′ direction, a regulatory sequence such as apromoter, a polynucleotide encoding a peptide of the present invention,and a transcriptional termination signal sequence functional in a hostcell. The promoter can be of any of the types discussed herein, forexample, a tissue specific promoter, a developmentally regulatedpromoter, an organelle specific promoter, a seed specific promoter, aplastid specific promoter, etc. The expression cassette can furthercomprise an operably linked targeting, transit, or secretion peptidecoding region capable of directing transport of the protein produced.The expression cassette can also further comprise a nucleotide sequenceencoding a selectable marker and/or a purification moiety.

[0068] More particularly, the present invention includes recombinantconstructs comprising an isolated polynucleotide sequence encoding theantifungal peptides of the present invention. The constructs can includea vector, such as a plasmid or viral vector, into which the sequence hasbeen inserted, either in the forward or reverse orientation. Therecombinant construct can further comprise regulatory sequences,including, for example, a promoter operatively linked to the sequence.Large numbers of suitable vectors and promoters are known to thoseskilled in the art and are commercially available.

[0069] A further embodiment of the present invention relates totransformed host cells containing constructs comprising theoligonucleotide sequences of the present invention. The host cell can bea higher eukaryotic cell, such as a mammalian or plant cell, or a lowereukaryotic cell such as a yeast cell, or the host can be a prokaryoticcell such as a bacterial cell. Introduction of the construct into thehost cell can be accomplished by a variety of methods including calciumphosphate transfection, DEAE-dextran mediated transfection, Polybrene,protoplast fusion, liposomes, direct microinjection into the nuclei,scrape loading, and electroporation. In plants, a variety of differentmethods can be employed to introduce transformation/expression vectorsinto plant protoplasts, cells, callus tissue, leaf discs, meristems,etc., to generate transgenic plants. These methods include, for example,Agrobacterium-mediated transformation, particle gun delivery,microinjection, electroporation, polyethylene glycol-mediated protoplasttransformation, liposome-mediated transformation, etc. (reviewed inPotrykus (1991) Annu. Rev. Plant Physiol. Plant Mol. Biol. 42: 205).

[0070] Peptides produced by expression of the polynucleotides of thepresent invention can be obtained by transforming a host cell by any ofthe previously described methods, growing the host cell underappropriate conditions, inducing expression of the polynucleotide andisolating the protein(s) of interest. If the protein in retained withinthe host cell, the protein can be obtained by lysis of the host cells,while if the protein is a secreted protein, it can be isolated from theculture medium. Several methods are available for purification ofproteins and are known to those of ordinary skill in the art. Theseinclude precipitation by, for example, ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography, lectinchromatography, high performance liquid chromatography (HPLC),electrophoresis under native or denaturing conditions, isoelectricfocusing, and immunoprecipitation.

[0071] Alternatively, peptides encoded by the polynucleotides of thepresent invention can be produced by chemical synthesis using eithersolid-phase peptide synthesis or by classical solution peptide synthesisalso known as liquid-phase peptide synthesis. In oligomer-supportedliquid phase synthesis, the growing product is attached to a largesoluble polymeric group. The product from each step of the synthesis canthen be separated from unreacted reactants based on the large differencein size between the relatively large polymer-attached product and theunreacted reactants. This permits reactions to take place in homogeneoussolutions, and eliminates tedious purification steps associated withtraditional liquid phase synthesis. Oligomer-supported liquid phasesynthesis has also been adapted to automatic liquid phase synthesis ofpeptides.

[0072] For solid-phase peptide synthesis, the procedure entails thesequential assembly of the appropriate amino acids into a peptide of adesired sequence while the end of the growing peptide is linked to aninsoluble support. Usually, the carboxyl terminus of the peptide islinked to a polymer from which it can be liberated upon treatment with acleavage reagent. In a common method, an amino acid is bound to a resinparticle, and the peptide generated in a stepwise manner by successiveadditions of protected amino acids to produce a chain of amino acids.Modifications of the technique described by Merrifield are commonly used(see, e.g., Merrifield, J. Am. Chem. Soc. 96: 2989-93, 1964). In anautomated solid-phase method, peptides are synthesized by loading thecarboxy-terminal amino acid onto an organic linker (e.g., PAM,4-oxymethylphenylacetamidomethyl), which is covalently attached to aninsoluble polystyrene resin cross-linked with divinyl benzene. Theterminal amine may be protected by blocking with t-butyloxycarbonyl.Hydroxyl- and carboxyl-groups are commonly protected by blocking withO-benzyl groups. Synthesis is accomplished in an automated peptidesynthesizer, a number of which are commercially available. Followingsynthesis, the product may be removed from the resin. The blockinggroups are removed typically by using hydrofluoric acid ortrifluoromethyl sulfonic acid according to established methods (e.g.,Bergot and McCurdy, Applied Biosystems Bulletin, 1987). Followingcleavage and purification, a yield of approximately 60 to 70% istypically produced. Purification of the product peptides is accomplishedby, for example, crystallizing the peptide from an organic solvent suchas methyl-butyl ether, then dissolving in distilled water, and usingdialysis (if the molecular weight of the subject peptide is greater thanabout 500 daltons) or reverse high-pressure liquid chromatography (e.g.,using a C.sup.18 column with 0.1% trifluoroacetic acid and acetonitrileas solvents) if the molecular weight of the peptide is less than 500daltons. Purified peptide may be lyophilized and stored in a dry stateuntil use. Analysis of the resulting peptides may be accomplished usingthe common methods of analytical high pressure liquid chromatography(HPLC) and electrospray mass spectrometry (ES-MS).

[0073] In general, transgenic plants comprising cells containingpolynucleotides of the present invention can be produced by any of theforegoing methods; selecting plant cells that have been transformed on aselective medium; regenerating plant cells that have been transformed toproduce differentiated plants; and selecting a transformed plant thatexpresses the protein(s) encoded by the polynucleotides of the presentinvention at a desired level. Specific methods for transforming a widevariety of dicots and obtaining transgenic plants are well documented inthe literature (Gasser and Fraley, Science 244:1293, 1989; Fisk andDandekar, Scientia Horticulturae 55:5, 1993; and the references citedtherein).

[0074] Successful transformation and plant regeneration have beenachieved in a variety of monocots. Specific examples are as follows:asparagus (Asparagus officinalis; Bytebier et al. (1987) Proc. Natl.Acad. Sci. USA 84: 5345); barley (Hordeum vulgarae; Wan and Lemaux(1994) Plant Physiol. 104: 37); maize (Zea mays; Rhodes et al. (1988)Science 240: 204; Gordon-Kamm et al. (1990) Plant Cell 2: 603; Fromm etal. (1990) Bio/Technology 8: 833; Koziel et al. (1993) Bio/Technology11: 194); oats (Avena sativa; Somers et al. (1992) Bio/Technology 10:1589); orchardgrass (Dactylis glomerata; Horn et al. (1988) Plant CellRep. 7: 469); rice (Oryza sativa, including indica and japonicavarieties; Toriyama et al. (1988) Bio/Technology 6: 10; Zhang et al.(1988) Plant Cell Rep. 7: 379; Luo and Wu (1988) Plant Mol. Biol. Rep.6: 165; Zhang and Wu (1988) Theor. Appl. Genet. 76: 835; Christou et al.(1991) Bio/Technology 9: 957); rye (Secale cereale; De la Pena et al.(1987) Nature 325: 274); sorghum (Sorghum bicolor; Cassas et al. (1993)Proc. Natl. Acad. Sci. USA 90: 11212); sugar cane (Saccharum spp.; Bowerand Birch (1992) Plant J. 2: 409); tall fescue (Festuca arundinacea;Wang et al. (1992) Bio/Technology 10: 691); turfgrass (Agrostispalustris; Zhong et al. (1993) Plant Cell Rep. 13: 1); and wheat(Triticum aestivum; Vasil et al. (1992) Bio/Technology 10: 667; Weeks etal. (1993) Plant Physiol. 102: 1077; Becker et al. (1994) Plant J. 5:299).

[0075] In one preferred embodiment, plants are transformed withrecombinant polynucleotides encoding the antifungal peptides of thepresent invention which result in the peptides being secreted by theplant. In another preferred embodiment, the antifungal peptides aresecreted by the roots of the transformed plant. Plants secretingantifungal peptides can be constructed by the above described methodsusing expression cassettes which incorporate a secretion sequence thatdirects secretion of the peptides. Alternatively, plants can betransformed with a nucleotide sequence encoding a fusion proteinconstructed from the antifungal peptides of the present invention and aprotein which is normally secreted by the plant. For example, a fusionprotein can be produced between an antifungal peptide and the cytokininoxidase enzyme. Cytokinin oxidase is a protective enzyme that acts todegrade exogenous cytokinins that could interfere with plant growthcontrol. By fusing the antifungal peptides to the region of thecytokinin oxidase gene controlling secretion, the antifungal peptidewould be secreted by the transformed plant, thus providing protectionfrom pathogenic fungi.

[0076] Before being used to transform plants, fusion proteins containingantifungal peptides can be screened for activity using the phage displaymethod of the present invention. In general, a fusion protein can beconstruction containing, an antifungal peptide; the secretory controlportion of a protein, such as cytokinin oxidase; and the pVIII or pIIIphage coat protein. Phage displayed fusion proteins so constructed canthen be screened using the method of the present invention to selectthose fusion proteins that bind to a target pathogenic fungus and resultin alternations which limit pathogenicity.

EXAMPLES

[0077] The following examples are intended to provide illustrations ofthe application of the present invention. The following examples are notintended to completely define or otherwise limit the scope of theinvention.

Example 1 Fungal Species and Zoospore Production

[0078] The fungal strains used were P. capsici (ATCC 15399); P. sojae(strain 7-6-1, race 25) (A. F. Schmitthenner, Ohio State University);and Phytophthora parasitica. All cultures were maintained as mycelia onlima bean agar plates (P. sojae) or corn meal agar plates (Difco, USA)(P. capsici and P. parasitica) at 15° C. Mycelium copies were made bytransferring plugs of mycelium (5 mm×5 mm) to agar plates containingclarified 10% V8® vegetable juice (Campbell Soup Co., USA). Three plugsper plate were grown for three to six days at 25° C. Sporangiaproduction was induced in P. capsici by trimming the plates andincubating at 25° C. with light. After one to two days, zoospore releasewas induced by flooding the plates with sterile water for 20 to 30minutes. P. parastica zoospore production was identical to that of P.capsici except that the plates were washed with sterile water for twominutes prior to incubating at 25° C. with light. Zoospore release wasinduced from P. sojae sporangia by flooding the plates four times insterile water at 30 minute intervals. Zoospores were released within twoto four hours. After their release, zoospores were filtered through fourlayers of cheesecloth to remove sporangial cases and mycelial fragments.A sample of the suspension was vortexed for 30 seconds to induceencystment and the cysts counted under a microscope in a hemacytometer.

Example 2 Preparation of Starved K91kan E. coli Cells and Titerinz Phageas Transducing Units

[0079] Prior to library screening, the phage were titered as tetracyclintransducing units (TU) in starved K91BluKan (kanomycin resistant)Escherichia coli cells, according to published methods (Smith & Scott,Methods in Enzymology, 217:228-257, 1993; Yu and Smith, Methods inEnzymology, 267:3-27, 1996). Transducing units are an effective way ofmeasuring the infectivity of the phage and are usually expressed asTU/ml of phage. In brief, K91BluKan cells were grown at 37° C. withvigorous shaking (˜170 rpm) in 20 ml superbroth (Smith and Scott,Methods in Enzymology, 217:228-257, 1993) to mid log phase (OD₆₀₀˜0.45).The cells were then incubated with gentle shaking for an additional 5minutes to allow any sheared F pIII to regenerate. The cells werecentrifuged in a sterile 50 ml Oak Ridge tube at 2,200 rpm for 10minutes in a Sorvall SS34 rotor at 4° C. The supernatant was poured offand the cells were resuspended in 20 ml of 80 mM NaCl, placed in a 125ml culture flask and shaken gently for 45 minutes at 37° C. and 70 rpm.The cells were then centrifuged as above and resuspended in 1 ml coldNAP buffer. The starved cells were stored at 4° C. and remainedinfective for 3 to 5 days.

[0080] The phage were titered as transducing units (TU) in E. coliK91BluKan starved cells (prepared as above). Phage were analyticallytitered using TBS/gelatin as the diluent. Ten microliters of each phagedilution were deposited as a droplet on the inner wall of a 15 mlsterile disposable tube held at a 10° angle from the horizontal. Tenmicroliters of starved E. coli K91BluKan cells were added to each phagedroplet and this was incubated for 10 minutes at room temperature toallow time for the phage to infect the concentrated cells. After 10minutes, 1 ml of superbroth containing 0.2 mg/ml tetracyclin was addedto the cells and incubated for 20 to 40 minutes at 37° C. with shaking.For amplification of the f88-4/15 mer phage, the superbroth alsocontained 1 mM IPTG to induce recombinant pVIII expression. The infectedcells were then spread (200 ml per plate) on Luria-Bertani (LB) platescontaining 40 mg/ml tetracyclin. The plates were then incubated for ˜24hr at 37° C.

Example 3 Selection of Zoospore Binding Phage

[0081] An aliquot of 10¹¹ transducing units (TU) from the f8-1 library(Petrinko et al., Protein Engineering, 9:797-801, 1996) was added to 10⁶freshly released P. capsici zoospores at room temperature in 4 ml of 50mM LiCl and incubated for 30 minutes at room temperature with gentleagitation. The same procedure was used for the f88-4 library except insome cases P. sojae zoospores were used (Soj clones). The zoosporescontaining the bound phage were washed 10 times in 150 μl of 50 mM LiCland centrifuged at 1000× g for 45 seconds to remove unbound phage. Afterthe tenth wash, the bound phage were eluted with 200 μl of elutionbuffer (0.1 N HCl, glycine sufficient to bring pH to 2.2, 1 mg/ml bovineserum albumin). The eluted phage were amplified by infection of starvedE. coli K91BluKan cells as described above. The amplified phage werethen purified by precipitation with polyethylene glycol as describedbelow, and resuspended in TBS buffer as described by Smith and Scott(Methods in Enzymology, 217:228-257, 1993). An aliquot of purified phagewas subsequently re-applied to freshly released zoospores, as describedabove for a total of three affinity purifications and two amplificationsteps. Selective enrichment of the zoospore-binding phage was monitoredby calculating the percent yield after each round of selection asdescribed in Smith and Scott (Methods in Enzymology, 217:228-257, 1993).This was done by calculating the total phage (expressed as transducingunits) that was applied to the zoospores and measuring the total outputof phage (as transducing units) that was recovered from the zoosporesand expressing the result as a percentage. The yield of phage elutedfrom the zoospores after each round of screening was between 10⁻⁴ to10⁻⁵% indicating that this procedure was successful in selecting forphage binding to the zoospores. The zoospores were intact and sphericalafter the washing steps, showing that little or no encystment hadoccurred during the selection process.

Example 4 Phage Purification

[0082]E. coli K91BluKan cells infected with phage were grown overnightin 20 ml superbroth (containing 40 mg/ml tetracyclin) at 37° C. and 170rpm. The culture was centrifuged to pellet the E. coli cells (containingphage) in a SS34 rotor for 10 min at 5,000× g. The supernatant wasremoved and placed in a new Oak Ridge tube and PEG/NaCl (16.7%polyethylene glycol/3.3 M NaCl) was added at a rate of 150 l per mlsupernatant to precipitate the phage. The phage were precipitatedovernight at 4° C. and then pelleted by centrifugation in a 50 ml OakRidge tube at 10,000 rpm for 20 min in a SS34 rotor. The pelleted phagewere resuspended in 1 ml of Tris-buffered saline (TBS). This was againre-precipitated by the addition of 150 ml PEG/NaCl and left overnight at4° C. The phage were pelleted by centrifugation in a bench topcentrifuge and the pellet was re-suspended in TBS.

Example 5 DNA Isolation, Sequencing and Analysis

[0083] DNA used for sequencing was isolated from individual phage clonesaccording to the method of Smith and Scott (Methods in Enzymology,217:228-257, 1993). Single-stranded DNA was sequenced from the 3′ endusing an ABI Prism 377 automated sequencer (Applied Biosystems, FosterCity, Calif.) following the manufacturer's protocol. The primer used forf8 clones was 5′-GGAGCCTTTAATTGTATCGG-3′ (SEQ ID NO: 2). The primer usedfor f88 clones was 5′-AGT AGC AGA AGC CTG AAG A-3′ (SEQ ID NO: 3).

[0084] DNA sequences were translated using the “translate” program ofthe ExPASy Molecular Biology Server (website http://www.expasv.ch/).Sequences were compared with nucleic acid and protein sequences storedin sequence databases (GenBank, EMBL, dbEST, SwissProt, PIR) usingstandard algorithms (i.e.) FASTA (Lipman and Person, Science,227:1435-1441, 1985) and BLAST (Altschul et al., J. Molecular Biol.,215:403-410, 1990) commands. Peptide sequences were aligned usingClustalW (Thompson et al., Nuc. Acid Res., 22:4673-4680, 1994) with aPAM250 weight table and the dendogram viewed using TreeView (Page,Computer Applic. Biosci., 12:357-358, 1996). The f8-mer DNA sequencesobtained coded for 19 predicted peptide sequences (Table 1). Themajority of the peptides contained amino acid residues that werepredicted to be strong α-helical formers (i.e. Glu, Ala and Leu) andα-helical breakers (i.e. Gly and Pro). Despite the lack of a commonmotif, the ClustalW multiple sequence alignment program was used tocluster similar peptides in the form of a dendogram. The dendogram,constructed from the aligned peptides, indicated that the f8-mer peptidesequences could be grouped into six broad family groups as depicted inFIG. 1 and Table 1. Selected sequences from the f88-4/15 mer library areshown in Table 2.

Example 6 Encystment Assay

[0085] Selected phage clones were isolated according to Smith and Scott(Methods in Enzymology, 217:228-257, 1993), and twice purified usingpolyethylene glycol as described above with the exception that phagewere resuspended in distilled water instead of TBS. The virionconcentration was calculated by measuring the absorbance at A₂₆₉ (Smithand Scott, Methods in Enzymology, 217:228-257, 1993). Water droplets ofapproximately 20 μl containing about 400 freshly released zoospores wereincubated with phage at two-fold serial dilutions so that they containedphage-bearing peptides at concentrations of either 1×10¹⁰, 5×10⁹,2.5×10⁹ or 1.25×10⁹ virion/μl of droplet. A negative control received nophage and was used to monitor the amount of spontaneous encystment inthe zoospore population. After a 20 minute incubation at roomtemperature, the number of encysted zoospores was counted using amicroscope at 100× magnification. The virion concentration of phage wascalculated according to Smith and Scott (Methods in Enzymology,217:228-257, 1993).

[0086] The effectiveness of the f8 peptides in inducing prematureencystment varied with sequence family and with phage concentration(FIG. 2). At a concentration of 1×10¹⁰ virion/μl (64 μM), many peptidefamilies were effective in inducing encystment. At a concentration of2.5×10⁹ virion, however, there was a 3- to 5-fold difference in thepeptides that caused high levels of encystment (Pc42, Pc78, Pc87 andPc64) and peptides causing low levels of encystment (Pc15B, Pc56 andPc45). At a concentration of 1.25×10⁹ (8 μM), all peptide families wereminimally effective in inducing encystment. The wild-type phage alsocaused encystment of the P. capsici zoospores; however, the fraction ofzoospores encysted by the selected phage was two to seven times greaterthan the fraction encysted by the wild-type phage in all experiments.The ability of P. capsici selected, phage-bearing peptides toprematurely encyst zoospores was specific for P. capsici. Little or noencystment was observed when P. sojae and P. parastica zoospores wereincubated with phage-bearing peptides at 1×10¹⁰ virion/μl, aconcentration that resulted in almost 100% encystment for P. capsicizoospores. Similar results were obtained with f88-4 15 mer peptides. Theability of representative 15 mer peptides to cause premature encystmentin comparison to the f-8 clone Pc87 is shown in FIG. 3.

Example 7 Binding Specificity

[0087] Phage-displayed peptides with high and low encystment inductionabilities were compared for their ability to bind to P. capsicizoospores. Phage clones Pc87 and Pc45 were randomly selected as therepresentative clones that induced high and low levels of encystment,respectively (cf. FIG. 2). Phage vector was included as a controltreatment. Phage clones were amplified by E. coli infections andpurified as described above. For each binding reaction, 5×10¹⁰ TU ofphage were incubated with 200,000 P. capsici zoospores. The bindingreaction and washes were performed as described in Example 3 for phageselection. Phage eluted from the zoospore population were titered in E.coli K91BluKan cells and expressed as total transducing units. A similarprocedure was used to determine whether the selected phage bound to P.capsici cysts.

[0088] Phage vector and clones Pc45 and Pc87 bound differentially to P.capsici zoospores. More than 10⁷ TU of phage Pc87 were eluted fromzoospores after 30 minutes incubation, while only about 50,000 TU ofphage Pc45 or phage vector were eluted under the same conditions (FIG.4). Moreover, binding was specific for the zoospore stage: less than 10⁴Pc87 TU were eluted from cysts-about the same background bindingobserved with control vector phage (FIG. 5).

Example 8 Construction of Secreted Fusion Protein

[0089] Carboxy-terminal DNA fusions were constructed to encode for thepeptides of interest by ligation of synthetic oligonucleotides into therestriction enzyme sites, HindIII and XbaI, of the carrier vector pJE-6.The plasmid vector pJE-6 was constructed from the Pichia pastorisexpression construct pROM-46 derived from the plasmid pPICZ-alpha(Invitrogen, Carlsbad, Calif.), previously described (Cregg et al.,Bio/Technology, 11:905-910, 1993; Rosenfeld, Methods in Enzymology,306:154-169, 1999). Plasmid pROM-46 was digested with restrictionendonuclease, HindIII, filled-in with Klenow enzyme and dNTP's, andre-ligated withT4 DNA ligase. These steps eliminated a HindIIIrestriction site present within the pPICZ-alpha plasmid sequence, andthe plasmid was designated pJE-4. The sequence at the 3′ end of thecoding sequence was mutagenized by PCR to replace the stop codon withthe restriction site HindIII. This plasmid was designated pJE-6.Synthetic oligonucleotides encoding for an exemplary peptide (Pc87,ADRPSMSPT, SEQ ID NO: 8), were ligated into the plasmid pJE-6, digestedwith HindIII and XbaI. This plasmid designated pJE-7 (FIG. 6). The pJE-7plasmid was sequenced to confirm the insert and the results are in shownin FIG. 7.

CONCLUSION

[0090] In light of the detailed description of the invention and theexamples presented above, it can be appreciated that the several aspectsof the invention are achieved.

[0091] It is to be understood that the present invention has beendescribed in detail by way of illustration and example in order toacquaint others skilled in the art with the invention, its principles,and its practical application. Particular formulations and processes ofthe present invention are not limited to the descriptions of thespecific embodiments presented, but rather the descriptions and examplesshould be viewed in terms of the claims that follow and theirequivalents. While some of the examples and descriptions above includesome conclusions about the way the invention may function, the inventorsdo not intend to be bound by those conclusions and functions, but putthem forth only as possible explanations.

[0092] It is to be further understood that the specific embodiments ofthe present invention as set forth are not intended as being exhaustiveor limiting of the invention, and that many alternatives, modifications,and variations will be apparent to those of ordinary skill in the art inlight of the foregoing examples and detailed description. Accordingly,this invention is intended to embrace all such alternatives,modifications, and variations that fall within the spirit and scope ofthe following claims. TABLE 1 FAMILY CLONE AMINO ACID SEQUENCE SEQ ID NO1   Pc56 AAPDLQDAM 4 2A Pc19 ADRLNSDAG 5 Pc36 ADRPSTTSL 6 Pc78 ADPPRTVST7 Pc87 ADRPSMSPT 8 Pc11 ADRTSNAST 9 2B Pc76 ADKSYIPSS 10 Pc65 AVRNPSHHS11 Pc44 ADPTPRGHS 12 Pc58 ADPTRQPHS 13 3A Pc45 AEHQNSAGP 14 Pc14ADARSAGAIS 15 Pc39 ADSKNAGPM 16 Pc53 AETKFSGSA 17 Pc15A ADPKGSGVT 18 3BPc15B AGLTSPNDM 19 Pc43/PC64 ADITDPMGA 20 4   PC29B AVGTHTPDS 21Pc12/Pc42 AVSPNVHDG 22

[0093] TABLE 2 CLONE AMINO ACID SEQUENCE SEQ ID NO. Cap1/18VAAFSLVWATHLMLS 23 Cap1/12 LTRCLVSTEMAARRP 24 Cap1/9 SAPYLPYFDLLHFPI 25Cap1/13 PSSYEASRRPEHWXF 26 Cap1/11 SATDTTLPMMTAIRS 27 Cap1/22TRLSPMESXAMLLAP 28 Cap1/20 LLPVSPPFAPNASST 29 Cap1/24 MSNFPTSHAPCPVEI 30Cap1/6 EFRKNYPSAAPLIPR 31 Cap1/23 PXVHGSIPLTPPLGF 32 Cap1/30LFXCYPPCTYSYCLS 33 Cap1/1 MSNFPTSHAPCPVXI 34 Cap1/16 PEWKSSWSPCTPRCP 35Cap1/28 AMSRWLRPRE(M/I)NAPP 36 Cap1/19 THTTFXVTVXLHEPP 37 Cap1/27MTSPRNSQLIVPFCL 38 Cap1/7 PTLGRFNRPSCSIIV 39 Soj2-2 APQCHPHLPFDMIHV 40Soj2-3 NHNSLPAQYLVXILR 41 Soj2-4; Soj2-6 DQPCTPSPDVSFYRS 42 Soj2-8VAAPSHWLKPSLDCF 43 Soj2-9 NPLYKNPPPRVAMCL 44 Soj2-19 LIFRYAPPPLFLRPP 45

[0094]

1 48 1 33 PRT Type 88 filamentous bacteriophage VARIANT (9)..(23) x=anyamino acid encoded by the codon NNK 1 Leu Val Pro Met Leu Ser Phe AlaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa XaaPro Ala Glu Gly Asp Asp Pro Ala Lys 20 25 30 Ala 2 20 DNA ArtificialSequence misc_feature (1)..(20) Primer 2 ggagccttta attgtatcgg 20 3 19DNA Artificial Sequence misc_feature (1)..(19) Primer 3 agtagcagaagcctgaaga 19 4 9 PRT Artificial Sequence DOMAIN (1)..(9) Random peptideinsert 4 Ala Ala Pro Asp Leu Gln Asp Ala Met 1 5 5 9 PRT ArtificialSequence DOMAIN (1)..(9) Random peptide insert 5 Ala Asp Arg Leu Asn SerAsp Ala Gly 1 5 6 9 PRT Artificial Sequence DOMAIN (1)..(9) Randompeptide insert 6 Ala Asp Arg Pro Ser Thr Thr Ser Leu 1 5 7 9 PRTArtificial Sequence DOMAIN (1)..(9) Random peptide insert 7 Ala Asp ProPro Arg Thr Val Ser Thr 1 5 8 9 PRT Artificial Sequence DOMAIN (1)..(9)Random peptide insert 8 Ala Asp Arg Pro Ser Met Ser Pro Thr 1 5 9 9 PRTArtificial Sequence DOMAIN (1)..(9) Random peptide insert 9 Ala Asp ArgThr Ser Asn Ala Ser Thr 1 5 10 9 PRT Artificial Sequence DOMAIN (1)..(9)Random peptide insert 10 Ala Asp Lys Ser Tyr Ile Pro Ser Ser 1 5 11 9PRT Artificial Sequence DOMAIN (1)..(9) Random peptide insert 11 Ala ValArg Asn Pro Ser His His Ser 1 5 12 9 PRT Artificial Sequence DOMAIN(1)..(9) Random peptide insert 12 Ala Asp Pro Thr Pro Arg Gly His Ser 15 13 9 PRT Artificial Sequence DOMAIN (1)..(9) Random peptide insert 13Ala Asp Pro Thr Arg Gln Pro His Ser 1 5 14 9 PRT Artificial SequenceDOMAIN (1)..(9) Random peptide insert 14 Ala Glu His Gln Asn Ser Ala GlyPro 1 5 15 10 PRT Artificial Sequence DOMAIN (1)..(10) Random peptideinsert 15 Ala Asp Ala Arg Ser Ala Gly Ala Ile Ser 1 5 10 16 9 PRTArtificial Sequence DOMAIN (1)..(9) Random peptide insert 16 Ala Asp SerLys Asn Ala Gly Pro Met 1 5 17 9 PRT Artificial Sequence DOMAIN (1)..(9)Random peptide insert 17 Ala Glu Thr Lys Phe Ser Gly Ser Ala 1 5 18 9PRT Artificial Sequence DOMAIN (1)..(9) Random peptide insert 18 Ala AspPro Lys Gly Ser Gly Val Thr 1 5 19 9 PRT Artificial Sequence DOMAIN(1)..(9) Random peptide insert 19 Ala Gly Leu Thr Ser Pro Asn Asp Met 15 20 9 PRT Artificial Sequence DOMAIN (1)..(9) Random peptide insert 20Ala Asp Ile Thr Asp Pro Met Gly Ala 1 5 21 9 PRT Artificial SequenceDOMAIN (1)..(9) Random peptide insert 21 Ala Val Gly Thr His Thr Pro AspSer 1 5 22 9 PRT Artificial Sequence DOMAIN (1)..(9) Random peptideinsert 22 Ala Val Ser Pro Asn Val His Asp Gly 1 5 23 15 PRT ArtificialSequence DOMAIN (1)..(15) Random peptide insert 23 Val Ala Ala Phe SerLeu Val Trp Ala Thr His Leu Met Leu Ser 1 5 10 15 24 15 PRT ArtificialSequence DOMAIN (1)..(15) Random peptide insert 24 Leu Thr Arg Cys LeuVal Ser Thr Glu Met Ala Ala Arg Arg Pro 1 5 10 15 25 15 PRT ArtificialSequence DOMAIN (1)..(15) Random peptide insert 25 Ser Ala Pro Tyr LeuPro Tyr Phe Asp Leu Leu His Phe Pro Ile 1 5 10 15 26 15 PRT ArtificialSequence DOMAIN (1)..(15) Random peptide insert 26 Pro Ser Ser Tyr GluAla Ser Arg Arg Pro Glu His Trp Xaa Phe 1 5 10 15 27 15 PRT ArtificialSequence DOMAIN (1)..(15) Random peptide insert 27 Ser Ala Thr Asp ThrThr Leu Pro Met Met Thr Ala Ile Arg Ser 1 5 10 15 28 15 PRT ArtificialSequence DOMAIN (1)..(15) Random peptide insert 28 Thr Arg Leu Ser ProMet Glu Ser Xaa Ala Met Leu Leu Ala Pro 1 5 10 15 29 15 PRT ArtificialSequence DOMAIN (1)..(15) Random peptide insert 29 Leu Leu Pro Val SerPro Pro Phe Ala Pro Asn Ala Ser Ser Thr 1 5 10 15 30 15 PRT ArtificialSequence DOMAIN (1)..(15) Random peptide insert 30 Met Ser Asn Phe ProThr Ser His Ala Pro Cys Pro Val Glu Ile 1 5 10 15 31 15 PRT ArtificialSequence DOMAIN (1)..(15) Random peptide insert 31 Glu Phe Arg Lys AsnTyr Pro Ser Ala Ala Pro Leu Ile Pro Arg 1 5 10 15 32 15 PRT ArtificialSequence DOMAIN (1)..(15) Random peptide insert 32 Pro Xaa Val His GlySer Ile Pro Leu Thr Pro Pro Leu Gly Phe 1 5 10 15 33 15 PRT ArtificialSequence DOMAIN (1)..(15) Random peptide insert 33 Leu Phe Xaa Cys TyrPro Pro Cys Thr Tyr Ser Tyr Cys Leu Ser 1 5 10 15 34 15 PRT ArtificialSequence DOMAIN (1)..(15) Random peptide insert 34 Met Ser Asn Phe ProThr Ser His Ala Pro Cys Pro Val Xaa Ile 1 5 10 15 35 15 PRT ArtificialSequence DOMAIN (1)..(15) Random peptide insert 35 Pro Glu Trp Lys SerSer Trp Ser Pro Cys Thr Pro Arg Cys Pro 1 5 10 15 36 15 PRT ArtificialSequence DOMAIN (1)..(15) Random peptide insert 36 Ala Met Ser Arg TrpLeu Arg Pro Arg Glu Xaa Asn Ala Pro Pro 1 5 10 15 37 15 PRT ArtificialSequence DOMAIN (1)..(15) Random peptide insert 37 Thr His Thr Thr PheXaa Val Thr Val Xaa Leu His Glu Pro Pro 1 5 10 15 38 15 PRT ArtificialSequence DOMAIN (1)..(15) Random peptide insert 38 Met Thr Ser Pro ArgAsn Ser Gln Leu Ile Val Pro Phe Cys Leu 1 5 10 15 39 15 PRT ArtificialSequence DOMAIN (1)..(15) Random peptide insert 39 Pro Thr Leu Gly ArgPhe Asn Arg Pro Ser Cys Ser Ile Ile Val 1 5 10 15 40 15 PRT ArtificialSequence DOMAIN (1)..(15) Random peptide insert 40 Ala Pro Gln Cys HisPro His Leu Pro Phe Asp Met Ile His Val 1 5 10 15 41 15 PRT ArtificialSequence DOMAIN (1)..(15) Random peptide insert 41 Asn His Asn Ser LeuPro Ala Gln Tyr Leu Val Xaa Ile Leu Arg 1 5 10 15 42 15 PRT ArtificialSequence DOMAIN (1)..(15) Random peptide insert 42 Asp Gln Pro Cys ThrPro Ser Pro Asp Val Ser Phe Tyr Arg Ser 1 5 10 15 43 15 PRT ArtificialSequence DOMAIN (1)..(15) Random peptide insert 43 Val Ala Ala Pro SerHis Trp Leu Lys Pro Ser Leu Asp Cys Phe 1 5 10 15 44 15 PRT ArtificialSequence DOMAIN (1)..(15) Random peptide insert 44 Asn Pro Leu Tyr LysAsn Pro Pro Pro Arg Val Ala Met Cys Leu 1 5 10 15 45 15 PRT ArtificialSequence DOMAIN (1)..(15) Random peptide insert 45 Leu Ile Phe Arg TyrAla Pro Pro Pro Leu Phe Leu Arg Pro Pro 1 5 10 15 46 36 DNA ArtificialSequence misc_feature (1)..(36) + strand of DNA encoding random peptidePc 87 46 agctagcaga tagaccatca atgtcaccaa catagt 36 47 36 DNA ArtificialSequence misc_feature (1)..(36) - strand of DNA encoding peptide Pc 8747 ctagactatg ttggtgacat tgatggtcta tctgct 36 48 611 PRT ArtificialSequence SIGNAL (1)..(85) Mat-alpha secretory sequence 48 Met Arg PhePro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala LeuAla Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln 20 25 30 Ile ProAla Asp Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe 35 40 45 Asp ValAla Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu 50 55 60 Phe IleAsn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val 65 70 75 80 SerLeu Glu Lys Arg Leu Ala Ala Gly Thr Pro Ala Leu Gly Asp Asp 85 90 95 ArgGly Arg Pro Trp Pro Ala Ser Leu Ala Ala Leu Ala Leu Asp Gly 100 105 110Lys Leu Arg Thr Asp Ser Asn Ala Thr Ala Ala Ala Ser Thr Asp Phe 115 120125 Gly Asn Ile Thr Ser Ala Leu Pro Ala Ala Val Leu Tyr Pro Ser Thr 130135 140 Gly Asp Leu Val Ala Leu Leu Ser Ala Ala Asn Ser Thr Pro Gly Trp145 150 155 160 Pro Tyr Thr Ile Ala Phe Arg Gly Arg Gly His Ser Leu MetGly Gln 165 170 175 Ala Phe Ala Pro Gly Gly Val Val Val Asn Met Ala SerLeu Gly Asp 180 185 190 Ala Ala Ala Pro Pro Arg Ile Asn Val Ser Ala AspGly Arg Tyr Val 195 200 205 Asp Ala Gly Gly Glu Gln Val Trp Ile Asp ValLeu Arg Ala Ser Leu 210 215 220 Ala Arg Gly Val Ala Pro Arg Ser Trp AsnAsp Tyr Leu Tyr Leu Thr 225 230 235 240 Val Gly Gly Thr Leu Ser Asn AlaGly Ile Ser Gly Gln Ala Phe Arg 245 250 255 His Gly Pro Gln Ile Ser AsnVal Leu Glu Met Asp Val Ile Thr Gly 260 265 270 His Gly Glu Met Val ThrCys Ser Lys Gln Leu Asn Ala Asp Leu Phe 275 280 285 Asp Ala Val Leu GlyGly Leu Gly Gln Phe Gly Val Ile Thr Arg Ala 290 295 300 Arg Ile Ala ValGlu Pro Ala Pro Ala Arg Ala Arg Trp Val Arg Phe 305 310 315 320 Val TyrThr Asp Phe Ala Ala Phe Ser Ala Asp Gln Glu Arg Leu Thr 325 330 335 AlaPro Arg Pro Gly Gly Gly Gly Ala Ser Phe Gly Pro Met Ser Tyr 340 345 350Val Glu Gly Ser Val Phe Val Asn Gln Ser Leu Ala Thr Asp Leu Ala 355 360365 Asn Thr Gly Phe Phe Thr Asp Ala Asp Val Ala Arg Ile Val Ala Leu 370375 380 Ala Gly Glu Arg Asn Ala Thr Thr Val Tyr Ser Ile Glu Ala Thr Leu385 390 395 400 Asn Tyr Asp Asn Ala Thr Ala Ala Ala Ala Ala Val Asp GlnGlu Leu 405 410 415 Ala Ser Val Leu Gly Thr Leu Ser Tyr Val Glu Gly PheAla Phe Gln 420 425 430 Arg Asp Val Ala Tyr Ala Ala Phe Leu Asp Arg ValHis Gly Glu Glu 435 440 445 Val Ala Leu Asn Lys Leu Gly Leu Trp Arg ValPro His Pro Trp Leu 450 455 460 Asn Met Phe Val Pro Arg Ser Arg Ile AlaAsp Phe Asp Arg Gly Val 465 470 475 480 Phe Lys Gly Ile Leu Gln Gly ThrAsp Ile Val Gly Pro Leu Ile Val 485 490 495 Tyr Pro Leu Asn Lys Ser MetTrp Asp Asp Gly Met Ser Ala Ala Thr 500 505 510 Pro Ser Glu Asp Val PheTyr Ala Val Ser Leu Leu Phe Ser Ser Val 515 520 525 Ala Pro Asn Asp LeuAla Arg Leu Gln Glu Gln Asn Arg Arg Ile Leu 530 535 540 Arg Phe Cys AspLeu Ala Gly Ile Gln Tyr Lys Thr Tyr Leu Ala Arg 545 550 555 560 His ThrAsp Arg Ser Asp Trp Val Arg His Phe Gly Ala Ala Lys Trp 565 570 575 AsnArg Phe Val Glu Met Lys Asn Lys Tyr Asp Pro Lys Arg Leu Leu 580 585 590Ser Pro Gly Gln Asp Ile Phe Asn Lys Leu Ala Asp Arg Pro Ser Met 595 600605 Ser Pro Thr 610

What is claimed is:
 1. A method for identification of non-immunoglobulinpeptides having an affinity for the surface of fungi comprising: (a)constructing a library of peptides by, (i) preparing randomoligonucleotides; (ii) inserting said oligonucleotides into anappropriate vector that expresses peptides encoded by said randomoligonucleotides on its surface and is capable of transfecting a hostcell; (iii) transfecting an appropriate host cell with said vector toamplify said vector in an infectious form to create a library ofpeptides on the surface of said vector; (b) contacting said vectorexpressing said peptide library with a target fungus and removingunbound vector; (c) eluting bound vector from said fungi; (d) amplifyingsaid bound vector; (e) sequencing the oligonucleotides contained in saideluted vector; (f) deducing the amino acid sequence of peptides encodedby said oligonucleotides contained in said eluted vector; and (g)selecting the non-immunoglobulin peptides.
 2. The method of claim 1,further comprising repeating steps (b) through (d) at least once.
 3. Themethod of claim 1, wherein said vector is a fusion phage vector.
 4. Themethod of claim 1, wherein said vector is a fusion phage vector selectedfrom the group consisting of type 8, type 88, type 8+8, type 3, type 33,type 3+3, type 6, type 66, type 6+6, phage T7 and phage
 8. 5. The methodof claim 1, wherein the sequence of said random oligonucleotide is GCAGNN (NNN)7 or SEQ ID NO:
 1. 6. The method of claim 1, wherein saidpeptide is expressed as part of a coat protein of said vector.
 7. Themethod of claim 6, wherein said coat protein is a pIII or a pVIII coatprotein.
 8. The method of claim 1, further comprising estimating thebinding affinity of said peptides to said target fungus.
 9. The methodof claim 1, wherein said peptides contain from 6 to 15 amino acids. 10.A composition comprising at least one substantially purified peptideselected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 31, SEQID NO: 33, and SEQ ID NO:
 4. 11. The composition of claim 10, whereinsaid composition is an antifungal composition.
 12. The composition ofclaim 11, wherein said composition alters the life cycle of members ofthe genus Phytophthora.
 13. The composition of claim 12, wherein saidcomposition alters the life cycle of Phytophthora capsici.
 14. Arecombinant polynucleotide comprising a sequence encoding a peptideselected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 31, SEQID NO: 33, and SEQ ID NO:
 4. 15. A recombinant vector comprising anucleotide sequence encoding a peptide selected from the groupconsisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 20, SEQ ID NO: 21,SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 31, SEQ ID NO: 33, and SEQ IDNO:
 4. 16. A cell transformed with the recombinant vector of claim 15.17. The cell of claim 16, wherein said cell is a plant cell.
 18. Anexpression cassette comprising as operatively linked components, apromoter, a nucleotide sequence of claim 14, and a transcriptiontermination signal sequence.
 19. The expression cassette of claim 18,further comprising an operatively linked secretion sequence.
 20. Theexpression cassette of claim 18, wherein said promoter is a tissuespecific promoter.
 21. The expression cassette of claim 18, wherein saidpromoter is a plant promoter.
 22. A transgenic plant comprising theexpression cassette of claim
 21. 23. A method for screening peptides forthe ability to affect development of a fungus comprising: (a)constructing a peptide library by, (i) preparing randomoligonucleotides; (ii) inserting said oligonucleotides into anappropriate vector that expresses peptides encoded by said randomoligonucleotides on its surface and is capable of transfecting a hostcell; (iii) transfecting an appropriate host cell with said vector toamplify said vector in an infectious form to create a library ofpeptides on said vector; (b) contacting said vector expressing saidpeptide library with a target fungus and removing unbound vector; (c)eluting bound vectors from said fungus; (d) amplifying said boundvectors; (e) isolating the oligonucleotides contained in said elutedvectors; (f) producing the peptides encoded by said oligonucleotidescontained in said eluted vectors; (g) contacting said peptides with atarget fungus; and (h) determining the effect of said peptides on saidfungus.
 24. The method of claim 23, further comprising repeating (b)through (d) at least once.
 25. The method of claim 23, wherein saidvector is a fusion phage vector.
 26. The method of claim 23, whereinsaid vector is a fusion phage vector selected from the group consistingof type 8, type 88, type 8+8, type 3, type 33, type 3+3, type 6, type66, type 6+6, phage T7 and phage
 8. 27. The method of claim 23, whereinthe sequence of said random oligonucleotide is GCA GNN (NNN)₇ or SEQ IDNO:
 1. 28. The method of claim 23, wherein said peptide is expressed aspart of a coat protein of said vector.
 29. The method of claim 28,wherein said coat protein is a pill or a pVIII coat protein.
 30. Themethod of claim 23, wherein said peptide is contacted with said targetfungus at different life stages of said fungus.
 31. The method of claim30, wherein said life stage is the zoospore life stage or the germlinglife stage.